The goal of the proposed research is to elucidate the mechanism of barium blockade of the selectivity filter of potassium channels and to determine the effects of barium blockade upon ion selectivity in the E71A mutant of the KcsA potassium channel. Since the selectivity filter of potassium channels is highly conserved, all knowledge gained from these studies is immediately applicable to a wide range of potassium channels. Voltage-activated potassium (K+) channels are membrane proteins that play a key role in the propagation of the nerve impulse. It is critical to their function that they maintain very high K+ o Na+ selectivity, but also conduct ions at a rate that approaches the diffusion limit. Currently, barium blockade electrophysiological experiments are one of the most reliable methods to determine ion selectivity in highly-selective potassium channels. However, little is known in detail about the positioning of the barium and potassium in the channels during these experiments or the effect of a barium blockade on the selectivity of the binding sites in the channel. Recently, and for the first time, experimental studies took place that investigated barium blockade in a potassium channel of known structure, the E71A mutant of KcsA. Slow inactivation is suppressed in the E71A mutant of KcsA, therefore that all information obtained about ion selectivity pertains to the conductive form of the selectivity filter. The existence of many structural studies of KcsA and additionally several studies of ion selectivity in KcsA make it uniquely suited for a detailed computational studies based on explicitly polarizable all-atom molecular dynamics simulations. Equilibrium and non-equilibrium molecular dynamics methods (free energy perturbation) will be used to determine the mechanism of barium blockade in the E71A mutant of the KcsA potassium channel and the effects of barium blockade on ion selectivity.